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Bartolomei Laboratory

Summary:

The research in my laboratory focuses on the study of genomic imprinting and X inactivation in mice.

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Organisms and Viruses

  • B6(Cast7) ( Mus musculus )

    This wildtype mouse line contains Mus musculus castaneus chromosome 7 on the C57BL/6 background and is used for allele-specific studies.

  • B6(CAST7P12X) ( Mus musculus )

    This wildtype mouse line contains Mus musculus castaneus chromosome 7, X, and part of 12 on the C57BL/6 background and is used in allele-specific studies.

  • Cast X ( Mus musculus )

    Cast X mice are mice with a Cast X chromosome on an otherwise mixed 129S1/SvImJ/Mus musculus castaneus EiJ background.

    129S1/SvImJ females were first mated to Mus musculus castaneus EiJ males and then F1 female progeny were mated to Mus musculus castaneus EiJ males. N2 progeny with a Cast X chromosome were identified. The N2 CastX females were then mated to 129S1/SvImJ males, to isolate additional CastX males. These CastX males (CastXm) were mated to N2 CastX females (CastXf) to maintain the Cast X mice.

  • Dnmt1c ( Mus musculus )

    This is a null DNA methyltransferase mutant from The Jackson Laboratory crossed with C57BL/6 or C57BL/6(CAST7).

  • Dnmt1n ( Mus musculus )

    This is a hypomorphic DNA methyltransferase mutant from The Jackson Laboratory crossed with C57BL/6.

  • H19AFP ( Mus musculus )

    Using gene targeting, a 2.5-kb region that contains the H19 endodermal enhancers was replaced with a fragment that harbors the Afp enhancers. The targeting vector was electroporated into E14 129 embryonic stem (ES) cells and neomycin-resistant clones were screened for a recombination event that introduced the Afp enhancer fragment and a cassette containing the neomycin resistance and thymidine kinase genes flanked by loxP sites (neoRtk) in place of the H19 endodermal enhancers (H19Afpneo). These cells were subsequently transfected with Cre recombinase to delete the neoRtk genes (H19Afp).

    Cells from targeted ES cell clones (with and without the neo r gene) were injected into C57BL/6J blastocysts, and the blastocysts were transferred to pseudopregnant female mice.

    Morula and sperm were preserved.

  • H19DMD-9CG ( Mus musculus )

    Nine of the ten CpGs in the DMD were eliminated by introducing point mutations into the four 21-bp repeats (R1-R4) by gene targeting. Embryonic day 14 ES were electroporated with the linearized targeting vector and clones were screened by G418 selection for a targeting event with a loxP-flanked neomycin resistance cassette. The neor cassette was removed with Cre-mediated recombination by transiently transfecting the targeted ES cells with pTurboCre plasmid. Cells from targeted ES cell clones were injected into C57BL/6J blastocysts, and the blastocysts were transferred to pseudopregnant female mice.

  • H19DMD-∆R ( Mus musculus )

    The four 21 bp repeats in the DMD that bind CTCF were deleted by gene targeting. Day 14 ES cells were electroporated with linearized targeting vector. Two correctly targeted independent clones were injected into C57BL/6 blastocysts and were then transferred to pseudopregnant female mice. The resulting chimeras were bred with C57BL/6. The neor cassette was removed by crossing male founders with females carrying a Cre recombinase gene under the control of the Zp3 promoter.

  • H19ICR-8nrCG ( Mus musculus )

    The H19ICR-8nrCG contains mutations in 8 CpGs at the ICR, which depletes 16% of CpGs without changing the size of the ICR or disrupting CTCF sites. Targeting vectors were linearized and electroporated into E14.1 ES cells. Correctly targeted ES cell clones were injected into C57BL/6J blastocysts and mice were generated by the Transgenic & Chimeric Mouse Facility at the University of Pennsylvania. Chimeras were obtained and mated to C57BL/6J mice. The neor cassette (flanked by loxP sites) was excised in the mouse by crossing heterozygous mutant mice (H19ICR-8nrCG/+) to mice expressing Cre recombinase under the control of the human cytomegalovirus promoter on a C57BL/6 genetic background (obtained from Edward Morissey).

  • H19ICR∆IVS ( Mus musculus )

    The H19ICR∆IVS deletion removes ~0.9 kb of sequence between CTCF sites 2 and 3 at the ICR, which reduces the size of the ICR by ~50% and deletes ~35% of the CpGs at the ICR. Targeting vectors were linearized and electroporated into E14.1 ES cells. Correctly targeted ES cell clones were injected into C57BL/6J blastocysts and mice were generated by the Transgenic & Chimeric Mouse Facility at the University of Pennsylvania. Chimeras were obtained and mated to C57BL/6J mice. The neor cassette (flanked by loxP sites) was excised by crossing heterozygous mutant mice (H19ICRΔIVSneo/+) to mice expressing Cre recombinase under the control of the human cytomegalovirus promoter on a C57BL/6J genetic background (obtained from Edward Morissey).

  • H19lxDMD ( Mus musculus )

    Using gene targeting, the 1.6-kb DMD sequence at the endogenous locus was flanked by loxP sites. The targeting vector was electroporated and clones were screened by G418 selction for a targeting event. Cells from targeted ES cell clones were injected into C57BL/6J blastocysts, and the blastocysts were transferred to pseudopregnant female mice. These cells were subsequently transfected with Cre recombinase to excise the PGK-neo cassette.

  • H19∆3.8kb-5'H19allele ( Mus musculus )

    Using gene targeting, 3.8 kb of sequence containing the entire DMD and G-rich repeat element was deleted. ES cells were electroporated with linearized targeting vector, and clones were screened by G418 selction for a targeting event. Cells from targeted ES cell clones were injected into C57BL/6J blastocysts, and the blastocysts were transferred to pseudopregnant female mice. Mice harboring the H19Δ3.8kb-5′H19neoR allele were mated to transgenic mice expressing Cre recombinase for germ line excision of the PGK-neo cassette.

    This line is maintained on C57BL/6 background.

  • H19∆4.2kb ( Mus musculus )

    Using gene targeting, the 4.2-kb domain between the end of the H19 transcription unit and the endodermal enhancers from a normally imprinted H19 transgene was removed in this mouse line. The targeting vector was electroporated and clones were screened by G418 selction for a targeting event that introduced a loxP site 5′ of the 4.2-kb region and the neoRtk cassette 3′ (H19Δ4.2neo). H19Δ4.2neo ES cells were subsequently electroporated with Cre and screened by Southern hybridization for deletion of the 4.2-kb domain and the neoRtk cassette, leaving one loxP site in this region (H19Δ4.2). Cells from targeted ES cell clones were injected into C57BL/6J blastocysts, and the blastocysts were transferred to pseudopregnant female mice.

  • H19∆DMD ( Mus musculus )

    ES cells were electroporated with linearized targeting vector, and clones were screened by G418 selection for a targeting event that replaced a region of H19 from approximately -2 to -4kb relative to the transcription start site (the DMD domain) with a loxP-flanked neomycin resistance cassette. Cre-mediated recombination removed the neomycin cassette. Cells from targeted ES cell clones were injected into C57BL/6J blastocysts, and the blastocysts were transferred to pseudopregnant female mice.

    Morula were preserved.

  • H19∆DMD∆G ( Mus musculus )

    Using gene targeting, 2.9 kb of sequence including most of the DMD including repeats R2-R4 and HS2, and the G-rich repeat element was deleted. ES cells were electroporated with linearized targeting vector, and clones were screened by G418 selction for a targeting event. Targeted clones were transiently transfected with a Cre recombinase-expressing plasmid for removal of the PGK-neo cassette. Cells from targeted ES cell clones were injected into C57BL/6J blastocysts, and the blastocysts were transferred to pseudopregnant female mice.

    Morula were preserved.

  • MBD1 ( Mus musculus )

    This line was generated by the group of Fred Gage at The Salk Institute for Biological Studies. Exons 2 through 10 were replaced by homologous recombination of a targeting vector containing lacZ and neo. The deleted region encoded the MBD1, CXXC1, CXXC2, and CXXC3 domains.

    LInearized targeting vector was electroporated into J1 embryonic stem cells. ES clones were injected into C57BL/6 blastocysts. MBD1+/- mice were generated by crossing chimeric mice with 129S4 (formerly 129/SvJae) females to generate MBD+/- F1 litters, which contain a 129S4 genetic background. Crossing F1 MBD+/- mice generated F2 MBD-/- mice.

  • MBD2 ( Mus musculus )

    This line was generated by the group of Adrian Bird. Exon 2 of the Mbd2 gene was replaced with the promoterless beta-geo cassette. The resulting transcript can encode the N-terminal 183 amino acids of MBD2, but translation then stops in the middle of the methyl-CpG binding domain.

    Gene targeting was performed in the ES cell line E14 TG2a, which is derived from the mouse substrain 129/Ola. ES cells were transfected with the linearized targeting vector by electroporation. Targeted ES cells were injected into C57BL/6 blastocysts. Chimeric pups were mated with C57BL/6 mice.

  • MBD3 ( Mus musculus )

    This line was generated by the group of Adrian Bird. Targeted deletion of Mbd3 was acheived by replacing a genomic fragment containing exons 2-7 of the Mbd3 gene with a promoterless beta-geo cassette in embryonic stem cells. This removed sequences encoding all but the N-terminal 36 amino acids of the protein.

    Gene targeting was performed in the ES cell line E14 TG2a, which is derived from the mouse substrain 129/Ola. ES cells were transfected with the linearized targeting vector by electroporation. Targeted ES cells were injected into C57BL/6 blastocysts. Chimeric pups were mated with C57BL/6 mice. The line is maintained by intercrossing heterozygotes.

  • RX1 ( Mus musculus )

    129S1/SvImJ females were first mated to Mus musculus castaneus EiJ males and then F1 female progeny were mated to Mus musculus castaneus EiJ or CastX males. Mice were bred to 129S1/SvImJ mice to maintain the existing recombinant X chromosome.

    Sperm was cryopreserved from RX1 mice.

  • RX2 ( Mus musculus )

    CastX females were mated to a male with a double recombinant X chromosome that was Mus musculus castaneus EiJ at distal ends and 129S1/SvImJ in the middle region. The founding double recombinant male was the offspring of a mating between a CastX male and a mixed background female heterozygous for Mus musculus castaneus EiJ and 129S1/SvImJ. Female progeny with a CastX and the double recombinant X chromosome were mated with CastX males to maintain existing and to generate new recombinant alleles.

    To maintain existing or to generate new recombinant X chromosomes, males with the recombinant X chromosome were crossed to CastX females and then female progeny were mated with CastX males.

    Sperm was cryopreserved from RX2 mice.

  • Zp3dsCTCF ( Mus musculus )

    Mice harbor a transgene that expresses an RNA hairpin designed to target CTCF mRMA under the control of a promoter specific to growing oocytes specific to growing oocytes [Zona pellucida 3].

  • Zp3dsMBD3 ( Mus musculus )

    DNA was injected into pronuclei of fertilized one-cell mouse eggs derived from (C57BL/6 x SJL)F1 intercrosses. Mice were maintained on a C57BL/6J background.

    In this mouse line, RNAi utilizes a transgene to reduce Mbd3 RNA levels. The transgene employs the zona pellucida 3 (Zp3) promoter which drives expression of linked sequences in growing oocytes. The promoter is upstream of an Mbd3 inverted repeat that generates an approximately 510-bp dsRNA.

  • ∆G ( Mus musculus )

    Using gene targeting, 1.3 kb of sequence including the G-rich repeat element was deleted. ES cells were electroporated with linearized targeting vector, and clones were screened by G418 selction for a targeting event. Targeted clones were transiently transfected with a Cre recombinase-expressing plasmid for removal of the PGK-neo cassette. Cells from targeted ES cell clones were injected into C57BL/6J blastocysts, and the blastocysts were transferred to pseudopregnant female mice.


Last updated: 2013-04-19T16:18:59.813-04:00

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The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016